1. Technical Field
The present invention relates to a method and reagents for performing a fluorescence polarization immunoassay (FPIA) to determine the presence or amount of phencyclidine and phencyclidine metabolites in samples, especially fluid biological samples such as urine, serum or plasma, to a method of making the reagents, and to an assay kit containing the reagents. The invention relates particularly to (1) reagents (tracers and antibodies, and an assay kit containing the tracers and antibodies) for determining the presence or amount of phencyclidine and phencyclidine metabolites in a sample; (2) immunogen compounds used to raise monoclonal or polyclonal antibodies; (3) synthetic methods for making the tracer and immunogen compounds; and (4) analytical methods for conducting the assay.
2. Background Art
Phencyclidine is a synthetic drug with potent analgesic and anesthetic properties. This drug has been shown to produce serious and prolonged post anesthetic confusion and delirium. Its tendency to produce hallucinations, euphoria, distortions in perceptions, and feelings of dissociation have lead to illicit use and abuse. Recurring abuse has intensified efforts to prevent its manufacture and distribution. Consistent with these efforts, there exists a need for detection methods which are rapid, reliable and selective for phencyclidine and phencyclidine metabolites.
Phencyclidine is metabolized into two major metabolites, 4-phenyl 4 piperidinocyclohexanol and 1 (1 phenylcyclohexyl) 4-hydroxypiperidine, each of which is excreted mostly in the urine along with the corresponding glucuronide conjugates. Detection of either phencyclidine or phencyclidine metabolites indicates phencyclidine use.
The biological fluid most frequently tested is urine. Urine samples are non invasive of the body, and are generally more accessible than blood samples. However, testing of other biological materials is also possible.
In the past, urine samples have been tested for the presence of phencyclidine and phencyclidine metabolites by thin layer chromatography (TLC), gas chromatoqraphy (GC) or high performance liquid chromatography (HPLC) assays. A significant disadvantage of each of these methods is that the assay time involved in these methods is typically lengthy.
In assays for phencyclidine, phencyclidine metabolites, and other substances, competitive binding immunoassays have provided a more satisfactory alternative. Typically, competitive binding immunoassays are used for measuring ligands in a test sample. (For purposes of this disclosure, a "ligand" is a substance of biological interest to be quantitatively determined by a competitive binding immunoassay technique). The ligands compete with a labeled reagent (a "ligand analog" or "tracer") for a limited number of ligand binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody, and the amount of ligand analog that will bind to the antibody is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations.
FPIA techniques provide a quantitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay. Such procedures are based on the principle that a fluorescent labeled compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Accordingly, when a tracer-antibody conjugate having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane polarized light, its rotation is much faster than the corresponding tracer antibody conjugate and the molecules become more randomly oriented. As a result, the light emitted from the unbound tracer molecules is depolarized.
A problem that heretofore has prevented the accurate determination of phencyclidine and other "drugs of abuse" in urine by FPIA techniques is that of riboflavin interference. Riboflavin, or vitamin B.sub.2, is a common constituent of many foods and of commercially available vitamin supplements. Riboflavin is excreted primarily in the urine and has a fluorescence spectrum quite similar to that of fluorescein. As a result, the presence of riboflavin in even moderate amounts in urine samples creates an interference which can produce erroneous data. While ordinary consumption of riboflavin is unlikely to produce more than trace amounts of riboflavin in the urine, test results can readily be distorted by the consumption of excessive quantities of vitamin supplements by persons wishing to prevent detection of phencyclidine.
The present invention offers an advance in the art in that tracers, a method for making the tracers, and an assay using the tracers and monoclonal or polyclonal antibodies are provided specifically for the determination of phencyclidines and phencyclidine metabolites without riboflavin interference.